Method for producing liposome and apparatus for producing liposome

ABSTRACT

Disclosed herein are a method for producing a miniaturized liposome on a large production scale, and an apparatus for producing a liposome which is to be used in the above-mentioned method. Provided is a method for producing a liposome, including a step of stirring a mixture containing an oil phase in which at least one lipid is dissolved in an organic solvent and a water phase in a first tank of an apparatus having the first tank and a circulation path, in which the ratio of the capacity of the circulation path to the total capacity of the tank and the circulation path is 0.4 or less and/or the time required for the mixture to return to the first tank after being discharged therefrom is within 2.0 minutes.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a division of co-pending U.S. patentapplication Ser. No. 15/625,391 filed Jun. 16, 2017, which is acontinuation of PCT International Application PCT/JP2015/085455 filed onDec. 18, 2015, which claims priority under 35 U.S.C. § 119 of JapanesePatent Application No. 257282/2014 filed on Dec. 19, 2014, all of whichare hereby expressly incorporated by reference into the presentapplication.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates to a method for producing a liposome andan apparatus for producing a liposome. Specifically, the presentinvention relates to a method for producing a liposome which can besuitably used for pharmaceutical applications, and an apparatus forproducing such a liposome.

2. Description of the Related Art

A liposome (hereinafter, also referred to as lipid vesicle) is a closedvesicle formed of a lipid bilayer membrane using lipids, and has anaqueous solution (outer water phase) in which liposomes are dispersedand a water phase (inner water phase) within the space of the closedvesicle. Liposomes have been studied for a variety of applications suchas immune sensors, artificial red blood cells, and carriers of drugdelivery systems taking advantage of the features such as barriercapacity, compound retention capacity, biocompatibility, the degree offreedom of setting the particle size, ready biodegradability, andsurface-modifying properties. As for the application of a carrier,liposomes can encapsulate water-soluble compounds, lipophiliclow-molecular weight and high-molecular weight materials, and a widerange of materials. In particular, in the case where the liposome isused as a carrier for a drug delivery system, the particle size of theliposome is preferably small from the viewpoint of permeability inbiological membranes.

Liposomes can be prepared by stirring and emulsifying a water phase andan oil phase in a stirring apparatus. Since foaming may occur during theemulsification in a stirring apparatus, it is preferable to have adefoaming means.

With regard to the defoaming means, for example, JP2008-302264Adiscloses a defoaming tank including a tank body for containing a liquidto be treated in a state in which a liquid surface is verticallyvariable and a plurality of defoaming nozzles for diffusing and ejectinga defoaming liquid from above the liquid to be treated, in which bubblescontained in the liquid to be treated are defoamed by spraying thedefoaming liquid from the plurality of defoaming nozzles toward theliquid surface of the liquid to be treated. It is also described thatthis defoaming tank is used in connection with a floating concentrationcolumn 6, which is an example of a solids floating apparatus.JP2006-297360A discloses a liquid circulation type reduced pressureconcentration apparatus consisting of a preheating reservoir tank for afoamable liquid, a defoaming machine and a condenser, which isconfigured to prevent foaming in such a way that a rotating body isplaced in the defoaming machine, the foamable liquid preheated in thepreheating reservoir tank is fed into the rotating body of the defoamingmachine, and the liquid droplets are finely dispersed into fineparticles and released from the rotating body. JP1995-328620A(JP-H07-328620A) discloses a centrifugal sedimentation type centrifugeapparatus for removing a solid from a mixture containing a liquid and asolid having a specific gravity smaller than that of the liquid, whichis characterized by providing a rotor having an inside disc. It is alsostated that a defoaming tank is provided. JP2000-027024A discloses anoil agent defoaming apparatus formed by connecting an oil agent tank andan oil agent application device to an oil agent delivery pipe and an oilagent return pipe, characterized in that a suction device having asuction port for sucking bubbles in the oil agent tank is provided suchthat the distance between the oil agent liquid surface in the oil agenttank and the suction port is 50 to 110 mm. JP2003-113596A discloses adefoaming apparatus having a function of sucking bubbles from a liquidsurface and breaking the bubbles by a centrifugal force of a rotatingbody and then returning the swollen liquid back to the liquid surface.

SUMMARY OF THE INVENTION

It has been attempted to reduce the loss of emulsification energy due tofoaming by providing a defoaming tank. Since emulsified oil droplets anddispersions are usually stable and do not undergo re-coalescence withina short period of time, a known combination of an emulsifying tank and adefoaming tank is configured to have a structure in which the size ofthe emulsifying tank is fixed to a minimum and the external tank isenlarged from the viewpoint of a scale-up suitability. That is, thecapacity of the defoaming tank becomes larger than the capacity of theemulsifying tank. However, in the method for producing a liposomeincluding a step of stirring a mixture containing an oil phase and awater phase, there is a problem that it is not possible to achieve thedesired miniaturization even if the emulsification time is prolongedbecause re-coalescence may occur due to retention in the emulsifyingtank.

In addition, the circulating flow rate between an emulsifying tank and adefoaming tank has not particularly been considered because theemulsified oil droplets and dispersions as described above are stable ingeneral emulsions. However, in the emulsification of liposomes, if thecirculation flow rate is small, the re-coalescence proceedscorresponding to the amount of the residence time in the circulationpath and therefore it has not been possible to achieve miniaturizationbeyond a certain level.

As described above, in the preparation of liposomes by an emulsificationmethod, until the solvent has been removed after emulsification, theminiaturized dispersion becomes an unstable equilibrium system in whichthe particle size is increased due to re-coalescence. Therefore, in thecase where the production scale becomes larger, only by simply extendingthe emulsification time, miniaturization is limited to a certainparticle size, and it cannot be further miniaturized. In order toachieve a predetermined miniaturization, it is necessary to increase theenergy per unit volume of the liquid supplied by an emulsifyingapparatus. However, if the size of the emulsifying apparatus is simplyenlarged to increase the output, air entrainment becomes severe, andtherefore most of the shearing energy is wasted due to shearing of airresulting in foaming, and consequently it is not possible to achieve apredetermined particle size.

It is an object of the present invention to provide a method forproducing a miniaturized liposome on large scale production, and anapparatus for producing a liposome which is to be used in theabove-mentioned method.

As a result of extensive studies to solve the above-mentioned problems,the present inventors have found that, in a method for producing aliposome, including a step of stirring a mixture containing an oil phaseand a water phase in a first tank of an apparatus having the first tankand a circulation path, it is capable of producing miniaturizedliposomes on a large production scale, by setting the ratio of thecapacity of the circulation path to the total capacity of the tank andthe circulation path to be 0.4 or less and/or setting the time requiredfor the mixture to return to the first tank after being dischargedtherefrom to be within 2.0 minutes. The present invention has beencompleted based on such findings. That is, according to the presentinvention, the following will be provided.

(1) A method for producing a liposome, comprising a step of stirring amixture containing an oil phase in which at least one lipid is dissolvedin an organic solvent and a water phase in a first tank of an apparatushaving the first tank and a circulation path, in which the ratio of thecapacity of the circulation path to the total capacity of the tank andthe circulation path is 0.4 or less and/or the time required for themixture to return to the first tank after being discharged therefrom iswithin 2.0 minutes.

(2) The method for producing a liposome according to (1), in which theapparatus has a second tank in the middle of the circulation path.

(3) The method for producing a liposome according to (2), in which theinside of the second tank has saturated vapor.

(4) The method for producing a liposome according to any one of (1) to(3), in which the first tank is filled with the mixture.

(5) The method for producing a liposome according to any one of (1) to(4), in which the mixture in the first tank is subjected to a pressureload.

(6) The method for producing a liposome according to any one of (1) to(5), in which the mixture is circulated in the first tank and thecirculation path using a diaphragm pump or a rotary pump.

(7) The method for producing a liposome according to any one of (1) to(6), in which the mixture is stirred in the first tank using anintermittent jet flow generating type emulsifying apparatus.

(8) The method for producing a liposome according to any one of (1) to(7), in which the lipid includes a polyethylene glycol-modified lipid.

(9) The method for producing a liposome according to any one of (1) to(8), in which the average particle size of the liposomes to be producedis 60 nm or less.

(10) The method for producing a liposome according to any one of (1) to(9), in which the capacity of the first tank is 10 L or more and 100 Lor less.

(11) The method for producing a liposome according to any one of (1) to(10), in which the ratio of the capacity of the circulation path to thetotal capacity of the tank and the circulation path is 0.4 or less andthe time required for the mixture to return to the first tank afterbeing discharged therefrom is within 2.0 minutes.

(12) An apparatus for producing a liposome, comprising a first tank anda circulation path, in which the ratio of the capacity of thecirculation path to the total capacity of the tank and the circulationpath is 0.4 or less.

(13) The apparatus for producing a liposome according to (12), furthercomprising a second tank in the middle of the circulation path.

(14) The apparatus for producing a liposome according to (12) or (13),further comprising a pump in the middle of the circulation path.

(15) The apparatus for producing a liposome according to any one of (12)to (14), in which the pump is a diaphragm pump or a rotary pump.

(16) The apparatus for producing a liposome according to any one of (12)to (15), in which an intermittent jet flow generating type emulsifyingapparatus is installed in the first tank.

According to the present invention, it is possible to produce aminiaturized liposome on a large production scale.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a configuration example of an apparatus for producing aliposome according to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “step” used herein includes not only an independent step, butalso a step which may not be clearly separated from another step,insofar as an expected effect of the step can be attained.

The numerical ranges shown with “to” in the present specification meansranges including the numerical values indicated before and after “to” asthe minimum and maximum values, respectively.

In referring herein to a content of a component in a composition, in acase where plural substances exist corresponding to a component in thecomposition, the content means, the total amount of the pluralsubstances existing within the composition, unless otherwise specified.

The present invention will be described in detail.

The method for producing a liposome according to the present inventionis a method for producing a liposome, including a step of stirring amixture containing an oil phase in which at least one lipid is dissolvedin an organic solvent and a water phase in a first tank of an apparatushaving the first tank and a circulation path, in which the ratio of thecapacity of the circulation path to the total capacity of the tank andthe circulation path is 0.4 or less and/or the time required for themixture to return to the first tank after being discharged therefrom iswithin 2.0 minutes.

In the present invention, it is possible to efficiently use the energyof the emulsifying apparatus for miniaturization of the liposomes, byproviding a circulation path preferably including a second tank(defoaming tank) outside the first tank (emulsifying tank) to therebysuppress the energy loss due to the shearing of air. Further, it ispossible to achieve the desired miniaturization since re-coalescence ofthe liposomes due to the residence can be suppressed by setting theratio of the capacity of the circulation path to the total capacity ofthe first tank and the circulation path to be 0.4 or less and/or there-coalescence of liposomes can be suppressed by setting the timerequired for the mixture to return to the first tank after beingdischarged therefrom to be within 2.0 minutes. From the viewpoint of thespecificity of liposomes that exist in an equilibrium system ofdispersion and re-coalescence, being capable of producing fine liposomesby producing liposomes under the above-mentioned conditions,particularly in the case where the production scale is large, is acompletely unexpected advantageous effect.

(Liposome)

Liposome is a lipid vesicle formed from a lipid bilayer containing lipidmolecules. Specifically, the liposome refers to a vesicle containing aclosed lipid having a space separated from the external environment by alipid bilayer formed based on the polarity of the hydrophobic groups andhydrophilic groups of the lipid molecules. The liposome is a closedvesicle formed of a lipid bilayer membrane using lipids, and has a waterphase (inner water phase) within the space of the closed vesicle. Theinner water phase contains water and the like. The liposome may besingle lamellar (monolayer lamellar, unilamellar, or single bilayermembrane) or multilayered lamellar (multilamellar, which is anonion-like structure having multiple bilayer membranes where individuallayers are compartmented by water-like layers). In the presentinvention, a monolayer lamellar liposome is preferred from the viewpointof safety and stability in pharmaceutical applications.

The liposome is preferably a liposome capable of encapsulating a drugand is not particularly limited in terms of form. The “encapsulating”means taking a form in which a drug is contained in an inner water phaseand a membrane with respect to the liposome. For example, the liposomemay be a form where a drug is encapsulated within a closed space formedof a membrane, a form where a drug is included in the membrane itself,or a combination thereof.

The size (average particle size) of the liposome is not particularlylimited and is preferably 100 nm or less, more preferably 30 to 70 nm,still more preferably 40 to 60 nm, and particularly preferably 40 to 55nm. The liposome is preferably in the form of a spherical shape or ashape close thereto. In the present invention, the “average particlesize” means a volume average value of the diameters of liposomes asmeasured by a dynamic light scattering method.

The component (membrane component) constituting the lipid bilayer of aliposome is selected from lipids. As the lipid, any one may be used aslong as it is dissolved in a mixed solvent of a water-soluble organicsolvent and an ester-based organic solvent. Specific examples of lipidsinclude phospholipids, lipids other than phospholipids, cholesterols andderivatives thereof. These components may be composed of single orplural components.

Examples of the phospholipid include natural or synthetic phospholipidssuch as phosphatidylcholine (lecithin), phosphatidyl glycerol,phosphatidic acid, phosphatidylethanolamine, phosphatidyl serine,phosphatidyl inositol, sphingomyelin, and cardiolipin, or hydrogenatedproducts thereof (for example, hydrogenated soybean phosphatidylcholine(HSPC)). Among these, preferred is a hydrogenated phospholipid such ashydrogenated soybean phosphatidylcholine, or sphingomyelin, and morepreferred is hydrogenated soybean phosphatidylcholine. In the presentinvention, the “phospholipid” also encompasses a phospholipid derivativein which the phospholipid is modified.

Lipids other than phospholipids may be lipids containing no phosphoricacid, and examples thereof include, but are not particularly limited to,glycerolipid which does not contain a phosphoric acid moiety within themolecule, and sphingolipid which does not contain a phosphoric acidmoiety within the molecule. In the present invention, the term “lipidsother than phospholipids” also encompasses derivatives of lipids otherthan phospholipids in which modifications have been made to the lipidsother than phospholipids.

In the case where the lipids other than phospholipids contain a basicfunctional group, for example, in the case where the lipids other thanphospholipids are a material where a compound having a basic functionalgroup is bonded to a lipid, the lipid is referred to as a cationizedlipid. The cationized lipid becomes capable of modifying, for example,the membrane of the liposome and therefore can enhance the adhesivenessto cells which are the target sites.

Examples of cholesterols include cholesterol which containscyclopentahydrophenanthrene as a basic skeleton whose carbon atoms arepartially or completely hydrogenated and derivatives thereof. Specificexamples of cholesterols include, but are not particularly limited to,cholesterol. When the average particle size decreases to 100 nm or less,the curvature of the lipid membrane becomes higher. Since thedeformation of the membrane arranged within the liposome also increases,a water-soluble drug becomes more susceptible to leakage. However, as ameans for suppressing leakage properties, it is effective to addcholesterol or the like in order to fill the deformation of the membranecaused by the lipid (membrane-stabilizing effect).

The addition of cholesterol into liposome compositions is expected tolower the fluidity of the liposome membrane by filling membrane gaps ofliposomes, or the like. Generally, it has been desired that the contentof cholesterol in liposomes is usually an amount of up to about 50 mol %of the total moles of lipid components (total lipid).

In the present invention, in total moles of lipid components in theliposome composition (total lipid contained in the liposomecomposition), the content of cholesterol is preferably 10 to 35 mol %,more preferably 15 to 25 mol %, and still more preferably 17 to 21 mol%.

In addition to the above-mentioned components, a hydrophilic polymer orthe like for improving retentivity in blood, fatty acid, diacetylphosphate or the like as a membrane structure stabilizer, orα-tocopherol or the like as an antioxidant may be added to the liposome.In the present invention, it is preferable not to use additives such asa dispersing aid not approved for use as an intravenous injection inpharmaceutical applications, for example, a surfactant or the like.

The liposome preferably contains hydrophilic polymer-modified productsof phospholipids, lipids other than phospholipids, or cholesterols asphospholipids, lipids other than phospholipids, cholesterols andderivatives thereof.

Examples of the hydrophilic polymer include, but are not particularlylimited to, polyethylene glycols, polyglycerols, polypropylene glycols,polyvinyl alcohols, a styrene-maleic anhydride alternating copolymer,polyvinylpyrrolidone, and synthetic polyamino acid. The above-mentionedhydrophilic polymers may be used alone or in combination of two or morethereof.

Among these, from the viewpoint of retentivity in blood of aformulation, preferred are polyethylene glycols, polyglycerols, andpolypropylene glycols, and more preferred are polyethylene glycol (PEG),polyglycerol (PG), and polypropylene glycol (PPG). Polyethylene glycol(PEG) is most commonly used and is preferable due to having an effect ofimproving the retentivity in blood.

The molecular weight of PEG is not particularly limited. The molecularweight of PEG is 500 to 10,000 daltons, preferably 1,000 to 7,000daltons, and more preferably 2,000 to 5,000 daltons.

In the liposome, it is preferable to use a lipid modified by PEG(PEG-modified lipid), together with the main lipid contained in theliposome. Specific examples of the PEG-modified lipid include1,2-distearoyl-3-phosphatidylethanolamine-PEG (DSPE-PEG; manufactured byNippon Oil & Fats Co., Ltd.; specifically1,2-distearoyl-3-phosphatidylethanolamine-PEG2000, or1,2-distearoyl-3-phosphatidylethanolamine-PEG5000 is preferable) anddistearoyl glycerol-PEG2000 (manufactured by Nippon Oil & Fats Co.,Ltd.). These PEG-modified lipids may be added in an amount of 0.3 to 50mass %, preferably 0.5 to 30 mass %, and more preferably 1 to 20 mass %with respect to total lipid content.

In the liposome, preferred is a lipid combination of hydrogenatedsoybean phosphatidylcholine (a main lipid contained in the liposome) and1,2-distearoyl-3-phosphatidylethanolamine-PEG (a lipid used incombination with the main lipid).

The liposome preferably does not contain an anionic polymer (polyanion).In the present invention, since it is possible to control the releaseability by means of an osmotic pressure of an inner water phase, thereare advantages in that general versatility is excellent, and drugs whichcan be used in liposomes are not limited.

(Method for Producing Liposome)

The method for producing a liposome according to the present inventionis a method for producing a liposome, including a step of stirring amixture containing an oil phase in which at least one lipid is dissolvedin an organic solvent and a water phase in a first tank of an apparatushaving the first tank and a circulation path, in which the ratio of thecapacity of the circulation path to the total capacity of the tank andthe circulation path is 0.4 or less and/or the time required for themixture to return to the first tank after being discharged therefrom iswithin 2.0 minutes.

Particularly preferred is a case satisfying both the condition in whichthe ratio of the capacity of the circulation path to the total capacityof the tank and the circulation path is 0.4 or less, and the conditionin which the time required for the mixture to return to the first tankafter being discharged therefrom is within 2.0 minutes.

The method for producing a liposome may include other steps such as anevaporation step of evaporating the organic solvent used in theemulsifying step, if necessary.

The emulsifying step of emulsifying mixed lipids dissolved in an organicsolvent to form a liposome, without a drying and solidifying step, isnot limited as long as it is a step of emulsification, but it ispreferably a step of applying a high shearing force and performingmicroparticulation in an emulsifying step including an organic solvent.If necessary, evaporation (desolvation) of the organic solvent used inthe emulsifying step may be carried out to form a liposome.

(Oil Phase)

As the organic solvent serving as an oil phase, a mixed solvent of awater-soluble organic solvent and an ester-based organic solvent isused. In the present invention, it is preferred that an organic solventsuch as chloroform, methylene chloride, hexane, or cyclohexane is notsubstantially used as the organic solvent, and it is more preferred thatthese organic solvents are not used at all.

The water-soluble organic solvent is not particularly limited, and it ispreferably an organic solvent having a property that is spontaneouslymiscible with water. Specific examples of the water-soluble organicsolvent include alcohols such as methanol, ethanol, n-propanol,isopropanol, n-butanol, isobutanol, and t-butanol; glycols such asglycerol, ethylene glycol, and propylene glycol; and polyalkyleneglycols such as polyethylene glycol. Among these, preferred arealcohols. The alcohol is preferably at least one selected from ethanol,methanol, 2-propanol, or t-butanol, more preferably at least oneselected from ethanol, 2-propanol, or t-butanol, and still morepreferably ethanol.

The ester-based organic solvent is not particularly limited, and it ispreferably an ester obtained from the reaction of organic acids andalcohols. Specifically, the ester-based organic solvent is preferably atleast one selected from ethyl acetate, methyl acetate, isopropylacetate, t-butyl acetate, or methyl propionate, more preferably ethylacetate, isopropyl acetate, or methyl propionate, and still morepreferably ethyl acetate.

The mixing ratio of water-soluble organic solvent:ester-based organicsolvent is not particularly limited, and it may be about 90:10 to 20:80and preferably about 70:30 to 25:75 by mass ratio. The mixed solvent ofa water-soluble organic solvent and an ester-based organic solvent mayfurther contain an aqueous solvent to be described below, such as wateror buffer. The aqueous solvent may be added in a range of, for example,1 to 30 mass %. The pH of the mixed solvent is not particularly limited,and it is in the range of preferably about 4 to 10 and more preferablyabout 6 to 9. The ester-based organic solvents may containphysiologically active substances or the like such as various drugswhich are soluble in these solvents.

The concentration of the lipid is not particularly limited and may beappropriately adjusted, but it may be 40 g/L to 250 g/L, preferably 100g/L to 200 g/L in terms of a solution where a mixed liquid of awater-soluble organic solvent and an ester-based organic solvent servesas a solvent.

(Water Phase)

As a water phase (aqueous solution) where the membrane components aredispersed when producing liposomes, water (distilled water, water forinjection, or the like), physiological saline, various buffers, anaqueous solution of sugars or a mixture thereof (aqueous solvent) ispreferably used. The buffer is not limited to organic and inorganicbuffer solutions, and a buffer having a buffering action in the vicinityof a pH close to that of a body fluid is preferably used and examplesthereof include phosphate buffer, Tris buffer, citrate buffer, acetatebuffer, and Good's buffer. The pH of the water phase is not particularlylimited, and it may be 5.0 to 9.0 and preferably 7.0 to 8.0. Forexample, a phosphate buffer (for example, pH=7.4) is preferably used.The aqueous solution finally contained in the inner water phase of theliposome may be an aqueous solution for dispersing the membranecomponents when producing the liposome, or may be water, physiologicalsaline, various buffers, an aqueous solution of sugars or a mixturethereof which is newly added. The water used as a water phase (aqueoussolution) is preferably free from impurities (dust, chemicals, or thelike).

(Emulsifying Step)

In the emulsifying step, an oil phase where at least one lipid has beendissolved in an organic solvent and a water phase are mixed to preparean aqueous solution containing lipids, which is then emulsified bystirring. An oil phase where lipids have been dissolved in an organicsolvent and a water phase are mixed, stirred and emulsified to therebyprepare an emulsion where an oil phase and a water phase are emulsifiedin an O/W type. After mixing, a liposome is formed by removing a portionor all of the organic solvent derived from the oil phase using anevaporating step to be described below. Alternatively, a portion or allof the organic solvent in the oil phase is evaporated during the courseof the stirring-emulsification to form a liposome.

As a method of stirring, ultrasonic waves or mechanical shearing forceis used for particle miniaturization. A preferred emulsifying apparatuswill be described later herein. In addition, extruder processing ofallowing to pass through a filter having a certain pore size ormicrofluidizer processing may be carried out for uniformity of theparticle sizes. Use of an extruder or the like can result in deformationof secondarily formed multivesicular liposomes into univesicularliposomes. In the present invention, it is preferred from the viewpointof simplification of the production process that a liposome of a statethat has no drug loading is used in the next step without extrusionprocessing.

In the present invention, the ratio of the capacity of the circulationpath to the total capacity of the first tank and the circulation path is0.4 or less and/or the time required for the mixture to return to thefirst tank after being discharged therefrom is within 2.0 minutes. Theratio of the capacity of the circulation path to the total capacity ofthe first tank and the circulation path is preferably 0.3 or less, morepreferably 0.20 or less, and an example thereof is 0.19. The lower limitof the ratio of the capacity of the circulation path to the totalcapacity of the first tank and the circulation path is not particularlylimited, but it is generally 0.10 or more.

The time required for the mixture to return to the first tank afterbeing discharged therefrom is preferably within 1.0 minute, morepreferably within 0.8 minutes, and still more preferably within 0.6minutes. The lower limit of the foregoing time is not particularlylimited, but it is generally 0.1 minutes or more from the viewpoint ofthe defoaming effects.

Emulsification is carried out by stirring in a first tank of anapparatus having the first tank and a circulation path. The capacity ofthe first tank is not particularly limited, but from the viewpoint oflarge scale production being favorable, it is preferably 10 L or moreand 100 L or less and more preferably 20 L or more and 100 L or less.

Preferably, the first tank is filled with a mixture. By fully fillingthe emulsifying tank, the gas-liquid interface is eliminated, and theloss of energy due to foaming can be further suppressed. If a smallamount of air remains in sight glass of the upper part of the firsttank, or the like, the effect is not affected, and the emulsifyingliquid part may be substantially filled with the mixture.

Preferably, the mixture in the first tank may be subjected to a pressureload. This makes it possible to further reduce the loss of energy due tofoaming. The pressure load can be realized, for example, by providing avalve narrowing the flow path in the middle of the circulation pathderived from the first tank.

In the present invention, a second tank for defoaming may be preferablyprovided in the middle of the circulation path. By providing the secondtank, defoaming can be performed efficiently. In the case where thecirculation path has the second tank, the above-mentioned capacity ofthe circulation path refers to the capacity of the entire circulationpath including the second tank. In addition, it is preferable that thesecond tank has saturated vapor from the viewpoint of suppressing coarsematerials being generated from skinning due to drying. Depending on theneeds, such as stabilizing the circulating flow rate, the second tankmay also be subjected to a pressure load. The pressure load may beachieved, for example, by introducing compressed air or compressednitrogen into the second tank.

When carrying out the emulsification, it is preferred to have a mixturecirculate in the first tank and the circulation path using a pump whichmay be a turbo-type pump (a centrifugal pump, a diagonal flow pump, oran axial flow pump), a positive displacement reciprocating pump (apiston pump, a plunger pump, or a diaphragm pump), a positivedisplacement rotary pump (a rotary pump, a gear pump, a vane pump, or ascrew pump), a tube pump, or the like. Among them, a tube pump or adiaphragm pump having no sliding surfaces may be preferably used. It isalso possible to preferably use a positive displacement rotary pump witha small pulsation of a circulating flow rate. Particularly preferred isa diaphragm pump or a rotary pump that can easily secure a large flowrate.

For emulsification, it is preferable to stir a mixture in the first tankusing an emulsifying apparatus. Examples of the emulsifying apparatusthat can be used include an impeller type, a sawtooth blade type, aclosed rotor type, a rotor/stator type, a static mixer type, a colloidmill type, and a high-pressure homogenizer type. Among them, preferredis an impeller type, a sawtooth type, a closed rotor type, or arotor/stator type which is suitable for batch processing in a tank.

Particularly preferred is a rotor/stator type which is capable ofgenerating a jet flow in a treatment liquid by high-speed rotation forminiaturization, and capable of sufficiently exerting shear forcesbetween liquid and liquid or liquid and wall surface. As an example, anintermittent jet flow generating type emulsifying apparatus can be used.

The intermittent jet flow generating type emulsifying apparatus iscomposed of a rotor and a screen, where the rotor rotates at a highspeed while keeping minute clearance with the screen. The velocity ofthe processing material that has been given kinetic energy through thehigh-speed rotating rotor is increased by being passed through the slitportion of the screen. The processing material with an increasedvelocity forms an intermittent jet flow within the processing materialin the tank and generates liquid-liquid shear forces at the velocityinterface. It is possible to carry out atomization by the shear forcesgenerated by this intermittent jet flow. The particle size and theparticle size distribution can be controlled by the combination of arotor and a screen.

In the present invention, an average particle size of a liposome to beprepared can be controlled by arbitrarily selecting the speed and timeof stirring. In view of obtaining a liposome having safety andstability, it is preferable to provide shearing at a circumferentialspeed of 15 m/sec or higher to an aqueous solution containing lipids.Shearing is not limited, and a specific example thereof is shearing at acircumferential speed of preferably 15 m/sec or higher and 35 m/sec orlower, more preferably shearing at a circumferential speed of 20 m/secor higher and 35 m/sec or lower, and still more preferably shearing at acircumferential speed of 23 m/sec or higher and 30 m/sec or lower.

(Apparatus for Producing Liposome)

The apparatus for producing a liposome according to the presentinvention includes a first tank and a circulation path, in which theratio of the capacity of the circulation path to the total capacity ofthe tank and circulation path is 0.3 or less. The preferred range of theabove-mentioned ratio is as described above in the presentspecification. Further, the preferred range of the capacity of the firsttank is also as described above in the present specification.

An example of the configuration of the apparatus for producing aliposome according to the present invention is shown in FIG. 1.

A first tank 1, which is an emulsifying tank, is provided with acirculation path 2. The liquid in the first tank 1 is discharged fromthe first tank 1 and circulates through the circulation path 2 to returnto the first tank 1. The first tank 1 is provided with an emulsifyingapparatus 3. By operating the emulsifying apparatus 3, the liquid isstirred and emulsified in the first tank 1. In the middle of thecirculation path 2, a second tank 4, which is a defoaming tank, and apump 5 are provided. The bubbles can be separated and removed in thesecond tank 4. In addition, the liquid can be circulated in thecirculation path by operating the pump 5. Further, the circulation flowrate can be controlled by adjusting the output of the pump.

(Evaporating Step)

In the present invention, an evaporating step may be provided ifnecessary. In the evaporating step, an organic solvent is evaporatedfrom the aqueous solution containing the liposomes obtained in theemulsifying step. In the present invention, the evaporating stepincludes at least one of a step of forcibly removing a portion or all ofthe organic solvent derived from the oil phase as an evaporating stepand a step of naturally evaporating a portion or all of the organicsolvent in the oil phase during the course of stirring-emulsification.

The method of evaporating an organic solvent in the evaporating step isnot particularly limited. For example, at least one of a step of heatingto evaporate an organic solvent, a step of continuing the standing orslow stirring after emulsification, or a step of performing vacuumdegassing may be carried out.

In the present invention, in the step of evaporating an organic solvent,it is preferred that the concentration of an organic solvent containedin an aqueous solution containing liposomes is to be 15 mass % or lesswithin 30 minutes since the start of a step of evaporating the organicsolvent.

A liquid temperature when carrying out the production method of thepresent invention can be appropriately adjusted, but the liquidtemperature at the time of mixing an oil phase and a water phase ispreferably higher than or equal to a phase transition temperature of thelipid to be used. For example, in the case where a lipid having a phasetransition temperature of 35° C. to 40° C. is used, the liquidtemperature is preferably set to 35° C. to 70° C.

The aqueous solution containing the liposomes prepared via anemulsifying step may be subjected to post-processing such ascentrifugation, ultrafiltration, dialysis, gel filtration, orfreeze-drying, for removal of components that had not been included inthe liposomes, or adjustment of a concentration and an osmotic pressure.

Particle sizes of the resulting liposomes can be made uniform by usingdialysis, filtration, extrusion processing, or the like. In the methodfor producing a liposome composition according to the present invention,it is preferred to prepare empty liposomes in a state where a drug isnot loaded, without subjecting to extrusion processing. Moreover, if itis desired to separate the drug encapsulated in liposomes from the drugnot encapsulated in liposomes, centrifugation, dialysis, gel filtration,or the like may be employed.

(Drug Loading Step)

The liposome obtained by the method of the present invention canencapsulate a drug. In the drug loading step of encapsulating the drug,in the case of encapsulating a water-soluble drug in the liposomes, thedrug can be encapsulated in the inner water phase of the liposome by amethod of dissolving the drug in an aqueous medium capable of performinghydration and swelling, followed by heating at a temperature higher thanor equal to the phase transition temperature, and sonication orextrusion. A drug may also be encapsulated in an inner water phase bydissolving the drug in the water phase at a time of lipidemulsification.

The drug contained in the liposome may be any water-soluble drug thatcan be encapsulated in liposomes, and specific examples thereof include,but are not limited to, water-soluble materials having a physiologicalactivity or a pharmacological activity such as enzymes, proteins,peptides, nucleic acids (DNA, mRNA, siRNA, miRNA), low-molecular weightcompounds, sugars (oligosaccharides and polysaccharides), polymercompounds, antitumor agents, antimicrobial agents, contrast agents,antioxidants, anti-inflammatory agents, whitening agents, humectants,and hair growing agents. In the case of using a liposome as a carrierfor a drug delivery system, the water-soluble drug is preferably alow-molecular weight compound from the viewpoint of stability.

Specific examples of the water-soluble drug include anticancer agentssuch as an anthracycline-based anticancer agent such as doxorubicin,daunorubicin or epirubicin, a cisplatin-based anticancer agent such ascisplatin or oxaliplatin, a taxane-based anticancer agent such aspaclitaxel or docetaxel, a vinca alkaloid-based anticancer agent such asvincristine or vinblastine, a bleomycin-based anticancer agent such asbleomycin, a sirolimus-based anticancer agent such as sirolimus, and ametabolic antagonist such as methotrexate, fluorouracil, gemcitabine,cytarabine, or pemetrexed. Among these, preferred is a water-solubledrug such as doxorubicin, gemcitabine, or pemetrexed.

The water-soluble drug encapsulated in the liposome is present in adissolved state in the inner water phase of the liposome. Here, withregard to the dissolved state, it is deemed to be encapsulated as adissolved state in a case where the amount of the drug filled withrespect to the volume of the liposome is below the saturation solubilityof the drug in the composition liquid of the inner water phase. Further,even when the amount of the drug filled is above the saturationsolubility of the drug, a case where drug crystals are not observed by acryo-transmission electron microscope (Cryo-TEM) and diffractionpatterns attributable to crystal lattice are not observed by X-raydiffraction (XRD) measurement indicates that most of the drug isdissolved due to acceleration of dissolution by a physicochemicalenvironment created by the lipid membrane, partial incorporation of thedrug into the lipid membrane or the like, and is deemed to beencapsulated as a dissolved state. In addition, a substance encapsulatedby a loading method in which a solid substance is formed inside theliposome and a drug is encapsulated is not in a dissolved state, evenwhen it is a highly water-soluble drug.

The water-soluble drug to be encapsulated in a dissolved statepreferably has a solubility in water of 1 mg/ml or more, and morepreferably a solubility in water of 10 mg/ml or more.

(Sterile Filtration)

In order to formulate an aqueous solution containing liposomes, obtainedby the method for producing a liposome composition according to thepresent invention, into a pharmaceutical composition, it is preferableto carry out sterile filtration. Regarding the filtration method, it ispossible to remove unwanted materials from the aqueous solutioncontaining liposomes by using a hollow fiber membrane, a reverse osmosismembrane, a membrane filter or the like. In the present invention, theaqueous solution containing liposomes is preferably filtered using afilter having a sterile pore size (preferably 0.2 μm sterile filter)although there is no particular limitation. Normally, adsorption oraggregation of liposomes onto a sterile filter may occur in thefiltration step. However, the present invention has unexpected effectssuch as little influence in pressure loss or the like when performingfiltration, since liposomes having a specific average particle size anda uniform particle size distribution are obtained.

To prevent an effect of deformation of liposomes on the average particlesize, the sterile filtration step and the below-described asepticfilling step are preferably carried out at a temperature lower than orequal to the phase transition temperature of the lipids constituting theliposome. For example, in the case where the phase transitiontemperature of the lipid is around 50° C., the sterile filtration stepand the below-described aseptic filling step are carried out at atemperature of preferably about 0° C. to 40° C., and more specificallyabout 5° C. to 30° C.

(Aseptic Filling)

The aqueous solution containing the liposomes obtained after sterilefiltration is preferably aseptically filled for medical applications.Known methods can be applied for aseptic filling. A liposome compositionsuitable for medical applications can be prepared by aseptically fillingthe liposome-containing aqueous solution in a container.

An aqueous solvent, an additive, or the like may be appropriately addedto the aqueous solution containing the liposomes obtained by the presentinvention to thereby prepare a pharmaceutical composition containingliposomes. In connection with the route of administration, thepharmaceutical composition may also contain at least one of a tonicityagent, a stabilizer, an antioxidant, or a pH adjusting agent which ispharmaceutically acceptable.

The tonicity agent is not particularly limited and examples thereofinclude inorganic salts such as sodium chloride, potassium chloride,sodium hydrogen phosphate, sodium dihydrogen phosphate, and potassiumdihydrogen phosphate; polyols such as glycerol, mannitol, and sorbitol;and sugars such as glucose, fructose, lactose, and sucrose.

The stabilizer is not particularly limited and examples thereof includesugars such as glycerol, mannitol, sorbitol, lactose, and sucrose.

The antioxidant is not particularly limited and examples thereof includeascorbic acid, uric acid, tocopherol homologues (for example, vitamin E,four tocopherol isomers α, β, γ, and δ), cysteine, and EDTA. Stabilizersand antioxidants may be respectively used alone or in combination of twoor more thereof.

Examples of the pH adjusting agent include sodium hydroxide, citricacid, acetic acid, triethanolamine, sodium hydrogen phosphate, sodiumdihydrogen phosphate, and potassium dihydrogen phosphate.

The pharmaceutical composition may contain an organic solvent, collagen,polyvinyl alcohol, polyvinyl pyrrolidone, a carboxyvinyl polymer, sodiumcarboxymethyl cellulose, sodium polyacrylate, sodium alginate,water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, casein, gelatin,agar, diglycerol, propylene glycol, polyethylene glycol, vaseline,paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA),mannitol, sorbitol, lactose, PBS, sodium chloride, sugars, abiodegradable polymer, a serum-free medium, each of which ispharmaceutically acceptable, or an additive which is acceptable as apharmaceutical additive.

In particular, in the context of the present invention, thepharmaceutical composition preferably contains ammonium sulfate,L-histidine, purified sucrose, sodium hydroxide, hydrochloric acid, orthe like.

The container in which a pharmaceutical composition is filled is notparticularly limited, and it is preferably made out of a material havinglow oxygen permeability. Examples of the container include a plasticcontainer, a glass container, and a bag made out of a laminate filmhaving an aluminium foil, an aluminium-deposited film, an aluminiumoxide-deposited film, a silicon oxide-deposited film, a polyvinylalcohol, an ethylene-vinyl alcohol copolymer, a polyethyleneterephthalate, a polyethylene naphthalate, a polyvinylidene chloride, orthe like as a gas barrier layer. If necessary, light may be shielded byadopting a bag or the like using a colored glass, an aluminium foil, analuminium-deposited film, or the like.

In the container in which a pharmaceutical composition is filled, inorder to prevent oxidation by oxygen present in the space of thecontainer, it is preferable to replace the gases in the container spaceand drug solution with inert gases such as nitrogen. For example, aninjection solution is bubbled with nitrogen, whereby the filling of theinjection solution into a container can be carried out under a nitrogenatmosphere.

The administration method of a pharmaceutical composition is preferablya parenteral administration. For example, intravenous injections such asintravenous drip, intramuscular injection, intraperitoneal injection,subcutaneous injection, intraocular injection, or intrathecal injectionmay be selected. The specific administration method of a liposomecomposition includes, for example, a syringe, and administration byintravenous drip.

The dose of a drug contained in the pharmaceutical composition isusually selected from a range of 0.01 mg to 100 mg/kg body weight/day.However, the liposome composition of the present invention is notlimited to such a dose.

EXAMPLES

Hereinafter, the present invention will be described in detail withreference to Examples. However, the present invention is not limited tosuch Examples. The mixing ratio in the solvent composition refers to avolume ratio. For example, “ethanol/ethyl acetate=90/10” refers to 90%ethanol/10% ethyl acetate by a volume ratio.

Example 1

a) Preparation of Emulsification Equipment

The emulsification equipment was prepared by combining a circulationpath (the capacity of the circulation path is 6 L) including anintermittent jet flow generating type emulsifying apparatus having arotating part with a slit outer diameter of 90 mm, an emulsifying tankwith a full-water capacity of 26 L, a defoaming tank, and a circulatingpump (direct-acting diaphragm pump) as shown in FIG. 1. The capacity ofthe circulation path is the capacity of the entire circulation pathincluding the defoaming tank.

b) Preparation of Oil Phase

Hydrogenated soybean phosphatidylcholine, cholesterol andN-(carbonyl-methoxypolyethylene glycol2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt(hereinafter referred to as DSPE-PEG) were mixed to a molar ratio of57/38/5, and then an organic solvent (ethanol/ethyl acetate=75/25) wasadded thereto, followed by warming to 70° C. and dissolution of thelipids to prepare an oil phase. Here, the oil phase was prepared suchthat the lipid concentration in the oil phase was 70 mmol/L.

c) Preparation of Water Phase

177 mmol/L of an aqueous ammonium sulfate solution was prepared and wasthen prepared as a water phase in an emulsifying tank.

d) Emulsification

The water phase prepared in c) was heated to 70° C. The oil phaseprepared in b) was added so that the volume ratio of water phase/oilphase=8/3 was obtained while operating an emulsifying apparatus at acircumferential speed of 15 m/s and operating a circulation pump at aflow rate of 20 L/min. The total amount of the liquid after the additionwas 32 L. After the emulsifying tank was fully hydrated, the overflowed6 L circulated through the circulation path including the defoamingtank. The internal pressure of the emulsifying tank was set to 30 kPa.In this state, the rotation of the emulsifying apparatus was increasedto a circumferential speed of 30 m/sec, followed by stirring for 30minutes.

e) Desolvation

The organic solvent was removed by ventilating air while heating theemulsion prepared in d) to a temperature higher than or equal to thephase transition temperature of the lipids, thereby producing liposomes.

Example 2

A liposome liquid was prepared in the same manner as in Example 1,except that the circulation pump was operated at a flow rate of 10L/min.

Example 3

A liposome liquid was prepared in the same manner as in Example 1,except that the circulation pump was operated at a flow rate of 5 L/min.

Comparative Example 1

A liposome liquid was prepared in the same manner as in Example 1,except that the circulation pump was operated at a flow rate of 2.5L/min.

Comparative Example 2

A liposome liquid was prepared in the same manner as in Example 1,except that the capacity of the emulsifying tank was set to 16 L, thecapacity of the circulation path was set to 16 L, and the circulationpump was operated at a flow rate of 12 L/min.

Example 4

A liposome liquid was prepared in the same manner as in Example 1,except that the capacity of the emulsifying tank was set to 16 L, thecapacity of the circulation path was set to 16 L, and the circulationpump was operated at a flow rate of 24 L/min.

Example 5

A liposome liquid was prepared in the same manner as in Example 1,except that the capacity of the emulsifying tank was set to 6 L, thecapacity of the circulation path was set to 26 L, and the circulationpump was operated at a flow rate of 32 L/min.

Example 6

A liposome liquid was prepared in the same manner as in Example 1,except that the internal pressure of the emulsifying tank was set at anatmospheric pressure and the circulation pump was operated at a flowrate of 10 L/min.

Example 7

A liposome liquid was prepared in the same manner as in Example 1,except that the capacity of the emulsifying tank was set to 20.8 L, thecapacity of the circulation path was set to 11.2 L, the liquid volume inthe emulsifying tank was set to 80% by volume, and the circulation pumpwas operated at a flow rate of 14 L/min.

Example 8

A liposome liquid was prepared in the same manner as in Example 1,except that the circulating pump was changed to a rotary pump.

(Measurement of Average Particle Size of Liposomes by Dynamic LightScattering Method)

The liposome liquids prepared in Examples 1 to 7 and Comparative Example1 were 40-fold diluted with pure water to obtain samples for measuringan average particle size. The average particle size of the samples formeasuring an average particle size was measured in terms of volumeaverage particle size using a particle size analyzer FPAR-1000AS(manufactured by Otsuka Electronics Co., Ltd.). The measurement resultsare shown in Table 1. Ratings were given as follows: A: average particlesize of 55 nm or less; B: average particle size of 56 nm or more and 60nm or less; and C: average particle size of 61 nm or more.

TABLE 1 Liquid volume Emulsifying Circulation Circulation CirculationAverage Emulsifying Circulation Total in emulsifying tank tank internalflow rate, time path/whole particle tank L path L L (volume ratio)pressure L/min (min) ratio size, nm Evaluation Example 1 26 6 32 Fullwater level 30 kPa 20 0.30 0.19 53 A (100%) Example 2 26 6 32 Full waterlevel 30 kPa 10 0.60 0.19 55 A (100%) Example 3 26 6 32 Full water level30 kPa 5 1.20 0.19 57 B (100%) Comparative 26 6 32 Full water level 30kPa 2.5 2.40 0.19 66 C Example 1 (100%) Comparative 16 16 32 Full waterlevel 30 kPa 12 1.33 0.50 65 C Example 2 (100%) Example 4 16 16 32 Fullwater level 30 kPa 24 0.67 0.50 56 B (100%) Example 5 6 26 32 Full waterlevel 30 kPa 32 0.81 0.81 58 B (100%) Example 6 26 6 32 Full water levelAtmospheric 10 0.60 0.19 57 B (100%) pressure Example 7 20.8 11.2 32 80%30 kPa 14 0.80 0.35 60 B Example 8 26 6 32 Full water level 30 kPa 200.30 0.19 53 A (100%)

As can be seen from Table 1, a small average particle size could beattained in Examples 1 to 7, which satisfy the conditions in which theratio of the capacity of the circulation path to the total capacity ofthe emulsifying tank and the circulation path is 0.4 or less and/or thecirculation time (the time required for the liquid to return to theemulsifying tank after being discharged therefrom) is within 2.0minutes, whereas a small average particle size could not be attained inComparative Example 1, which does not satisfy the above conditions.

(Preparation of Drug-Encapsulated Liposomes)

With respect to the non-drug encapsulated liposomes prepared in Example1, the lipid concentration of the liposome liquid was concentrated to arange of 120 to 150 mmol/L by tangential flow filtration, while theouter water phase of the liposome was replaced with a 0.09 mass %aqueous sodium chloride solution. A drug solution prepared by heating todissolve gemcitabine hydrochloride in a phosphate buffer solution wasadded thereto. The mixture was heated at about 70° C. for 30 minutes andthen cooled to room temperature. Thereafter, the drug liposome mixturewas filtered through a sterilization filter having a pore size of 0.2μm. Further, the drug liposome mixture was purified by dialysis againsta 9.4 mass % sucrose/10 mmol/L-histidine aqueous solution by tangentialflow filtration to remove non-encapsulated gemcitabine. This wasfollowed by filtration of liposomes through a sterilizing filter havinga pore size of 0.2 μm to obtain a solution of sterilegemcitabine-encapsulated liposomes having an average particle size of 65nm. According to the present invention, it was confirmed that liposomeshaving a single dispersion peak and a small average particle size of 100nm or less usable for medical applications can be produced. Although theaverage particle size of liposomes described in Evaluation of Table 1 ismeasured for empty liposomes, if the average particle size ofdrug-encapsulated liposomes is 100 nm or less, such liposomes can beused for medical use, with the average particle size of 65 nm being agood result.

INDUSTRIAL APPLICABILITY

According to the present invention, miniaturized liposomes can beproduced on large scale production. The liposomes produced by thepresent invention can be applied to medicines, cosmetics, foods, or thelike, and are particularly useful for medical applications.

EXPLANATION OF REFERENCES

-   -   1: first tank    -   2: circulation path    -   3: emulsifying apparatus    -   4: second tank    -   5: pump

What is claimed is:
 1. An apparatus for producing a liposome,comprising: a first tank; and a circulation path, wherein the ratio ofthe capacity of the circulation path to the total capacity of the tankand the circulation path is 0.4 or less.
 2. The apparatus for producinga liposome according to claim 1, further comprising a second tank in themiddle of the circulation path.
 3. The apparatus for producing aliposome according to claim 1, further comprising a pump in the middleof the circulation path.
 4. The apparatus for producing a liposomeaccording to claim 3, wherein the pump is a diaphragm pump or a rotarypump.
 5. The apparatus for producing a liposome according to claim 1,wherein an intermittent jet flow generating type emulsifying apparatusis installed in the first tank.
 6. The apparatus for producing aliposome according to claim 2, wherein an intermittent jet flowgenerating type emulsifying apparatus is installed in the first tank.